[Application effects of programmed cell death protein 1 antibody-internalizing Arg-Gly-Asp peptide conjugate combined with adoptive T cell therapy in cold tumors].

Objective: To explore how the programmed cell death protein 1(PD-1) antibody-internalizing Arg-Gly-Asp peptide conjugate [αPD-1-(iRGD)2], combined with adoptive T cell therapy (ACT), modulates the cold tumor microenvironment and exerts synergistic anti-tumor effects. Methods: αPD-1-(iRGD)2 was synthesized with glycosylation engineering. The penetrability and tumor-cytotoxicity enhancement of αPD-1-(iRGD)2 towards T cells were analyzed through flow cytometry in both conventional and 3D cell culture conditions for HGC27 cells, divided into the control group, the PD-1 monoclonal antibody monotherapy group (αPD-1 group), the PD-1 monoclonal antibody combined with free iRGD peptide group (αPD-1 iRGD group), and the conjugated drug group [αPD-1-(iRGD)2 group]. The biomarkers of T cell activation (CD25 and CD69) and effector function (GZMB、IFN-γ) were also characterized by flow cytometry. By establishing a classic"cold tumor"model- the BALB/c mouse 4T1 subcutaneous breast tumor model-using the same grouping as above, the antitumor effect of αPD-1-(iRGD)2 combined with ACT was evaluated, and flow cytometry was performed to analyze changes in the tumor microenvironment, including T cells, macrophages, and other immune components within the tumor, lymph nodes, and spleen. Fluorescence-labeled T cells were tracked in vivo via real-time near-infrared fluorescence imaging system. Flow cytometric analysis was conducted to characterize phenotypic changes in tumor-infiltrating lymphocyte (TIL) and to quantify TIM3 expression as an indicator of T cell exhaustion. Results: αPD-1-(iRGD)2 significantly enhanced the ability of T cells to penetrate HGC27 tumor spheroids, with the fluorescence intensity at the spheroid center increasing compared with the αPD-1 group at 30 min (P=0.008). The cytotoxicity of T cells in the conventional and 3D cell culture conditions for HGC27 cells was higher than that of the control group (all P<0.05). The competitive inhibition assays of T cell activation mediated by αPD-1-(iRGD)2 revealed that the proportions of GZMB⁺, IFN-γ⁺, and CD25⁺CD69⁺ T cells were all significantly elevated in the αPD-1-(iRGD)2 group compared with the control (all P<0.05). In the 4T1 subcutaneous breast tumor mouse model, combined treatment with αPD-1-(iRGD)2 and ACT reduced tumor volume to the control group (P<0.001), while the number of intratumoral CD8⁺ T cells increased compared with the αPD-1 group (P=0.001). The proportions of GZMB⁺ and IFN-γ⁺ CD8⁺ T cells rose to 61.98%±1.80% and 58.70%±2.15%, respectively (both P<0.05). In vivo near-infrared imaging further confirmed that the fluorescence intensity in tumor regions was higher in the αPD-1-(iRGD)2 group than in the αPD-1 group (all P<0.05). Phenotypic analysis showed that, compared with αPD-1 treatment, αPD-1-(iRGD)2 significantly reduced the proportion of TIM3⁺ exhausted T cells (all P<0.05) and increased the frequency of CD44⁺CD62L⁺ memory-phenotype T cells (all P<0.05). Conclusion: αPD-1-(iRGD)2 enhances the immune response of cold tumors to ACT by promoting intratumoral-infiltration of transferred T cells, alleviating the immunosuppressive microenvironment, and preserving the effector memory phenotype of T cells.